RU2753856C1 - Method for detecting antibodies in biomaterial using glass microstructure waveguides - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to biotechnology, in particular to a method for detecting antibodies. A method for detecting antibodies in a biomaterial using glass microstructure waveguides involves obtaining an optical immunosensor by introducing a reaction mixture of an analyzed sample with an antigen into the hollow core of a glass microstructure waveguide, followed by determining antibodies by the position of local maxima of the transmission spectrum of the sample, in real time, before and after filling with the mixture antigen and the analyzed solution containing the desired antibodies to this antigen.

EFFECT: ability to determine the presence of antibodies in biological material with high accuracy in real time.

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Description

The invention relates to biotechnology, namely to a technology for the determination of specific antibodies using glass microstructured waveguides with a hollow core (SMW PS) as optical immunosensors, and relates to a method for detecting the presence of analyzed antibodies in a test sample in real time. The invention can be used in the development of rapid methods for accounting for immune complexes and the creation of sensitive, selective and stable enzymeless sensors for direct detection of antibodies and antigens, as well as a variety of biomolecules and their conjugates.

Currently, there is a high degree of demand in medicine and veterinary medicine systems for the rapid determination of the antigen-antibody system, as well as the need to improve existing methods of serodiagnostics [Nakano S., Tsukimura T., Togawa T., Ohashi T., Kobayashi M., Takayama K. ., Kobayashi Y., Abiko H., Satou M., Nakahata T. Rapid immunochromatographic detection of serum anti-α-galactosidase A antibodies in fabry patients after enzyme replacement therapy. 2015 PloSOne. V. 10, no. 6. P. e0128351]. Immunochemical test systems are widely used to detect both infectious agent antigens and specific antibodies in biomaterials. The most common methods are traditionally enzyme-linked immunosorbent assay, immunofluorescence analysis, and immunoagglutination. In parallel, the technology for analyzing complex biological systems using biological microchips (biochips) is being developed. A popular analytical tool is biological microchips containing ordered biomolecular probes attached to the surface of a carrier such as glass, plastic, metal, etc. In the work [Utkin DV, Kireev MN, Guseva NP, Kaplun G.A., Kuklev V.E., Osina N.A. Development of a biological microchip to detect antibodies to antigens of the plague pathogen. 2019. Infection and Immunity. T. 9 (2), 393-398] describes the composition of the protein immunochip, which contains several antigenic markers, which allows you to identify a greater percentage of seroconversion than when using enzyme immunoassay.

Patent RU 2528099 proposes a biological microchip for detecting and multivariate analysis of anti-cholera antibodies in human blood serum, containing an array of V. cholerae O-antigens and cholera toxin discretely applied and immobilized on the substrate surface, grouped into separate zones. The invention provides for a parallel immunological analysis of several samples of human blood serum for the presence of anti-cholera antibodies to a wide range of antigens of the causative agent of cholera with the determination of class G and M immunoglobulins in a short time. The disadvantages of the method include the need to use an immunofluorescent label and species-specific secondary antibodies and the multistage analysis.

In most experimental works devoted to the study of antigen-antibody binding, the equilibrium characteristic is traditionally determined - the equilibrium constant of the antigen-antibody complex. The kinetic approach is used much less frequently, although it is more informative. This is due to methodological difficulties in obtaining and analyzing kinetic curves. In recent years, with the development of new research methods, more and more works devoted to the study of the kinetics of the formation of antigen-antibody complexes have appeared. The most common method for studying the kinetics of antigen binding to an antibody on the surface of single cells is flow cytometry [Schulz A.R., Baumgart S., Schulze J., Urbicht M., Grützkau A., Mei H.E. Stabilizing Antibody Cocktails for Mass Cytometry 2019 Cytometry A. No. 95 (8), 910-916]. The disadvantages of the method include the complexity of the technical equipment and the need for special preparation of the sample: it is labeled with special fluorescent dyes (fluorochromes).

The improvement of serodiagnostic tools is largely based on the development of the concept of biosensors - portable devices that attract with their mobility, availability, low cost and are intended for screening biologically important components, incl. antibodies. The overwhelming number of works aimed at creating biosensors is devoted to electrochemical methods of signal registration [Higson S. Biosensors for medical applications. 2012 Woodhead Publishing Limited. 337 p.]. They offer a number of advantages such as low manufacturing costs, simple design, reliable measurement, achievable low detection limits and small operating volumes. The latter is especially important when analyzing biological samples [Tian K., Prestgard M., Tiwar A. A review of recent advances in nonenzymatic glucose sensors. 2014. Materials Science and Engineering V. 41. P. 100-118]. The disadvantages of electrochemical biosensors are associated with the temperature and time instability of the biochemical receptors used, their high cost, and the need to introduce additional reagents and signal-forming substances into the analyzed solution.

Optical registration methods, like electrochemical ones, mainly detect immune complexes using secondary antibodies labeled with colloidal gold nanoparticles [Spitsyn A.N., Utkin D.V., Kireev M.N., Sharapova N.A., Erokhin P. S., Germanchuk V.G., Kochubei V.I. Optical registration of the formation of immune complexes using colloidal gold nanoparticles. 2018. Optics and Spectroscopy. T. 125. No. 5. S. 716-720]. A biological sensor is described, as well as a method for creating a biological sensor based on the use of surface plasmon resonance (patent RU 2527699) related to an optical phenomenon. The method allows the analysis of interactions in real time by registering the properties and changes in the properties of the test substance on the matrix. This method has the possibility of label-free biodetection, that is, without the use of radioactive or fluorescent labels. The disadvantages of the plasmon resonance method are the complexity of creating a multilayer structure for selective chemical interaction only with certain biological molecules of the analyte, optical interference, and the need to use photodetectors.

The most promising, in our opinion, is the creation of sensitive, selective and enzymeless optical sensors for direct detection of antibodies and antigens. One such example is a method (patent RU 2315313), according to which the determination of autoantibodies is carried out by changing the vibration frequency of a piezoelectric quartz resonator based on volume acoustic waves. The disadvantage of this method is the complexity, multistage and duration of the analysis.

WO 2013090177 discloses optical filtering methods using an SPPC resonator with drop resonators.

The known method of labelless multiple probing of analytes, biosensing and diagnostic analysis (US patent 20130005604).

An analysis of the prior art carried out by the applicant, including a search for patents and scientific and technical sources of information, did not reveal information about methods for detecting immune complexes, in particular antibodies, using biosensors based on glass microstructured waveguides with a hollow core (SMW PS).

The objective of the invention is to develop a method that allows in a short time with high accuracy to determine the presence of antibodies in biological material using a multifunctional sensor platform based on microstructural waveguides, combining the functions of a micro- and nano-reactor and an optical signal converter, in real time on standard equipment to measure the transmission spectra of the SSWS without additional costs for additional equipment and labor resources.

The technical result consists in the implementation of the specified purpose.

The technical result is achieved by a method for detecting antibodies in a biomaterial using glass microstructural waveguides, which provides for the production of an optical immunosensor by introducing a reaction mixture with a specific antigen and an analyzed sample into the structural shell of a glass microstructural waveguide (SMW PS), followed by determining the presence of antibodies by the difference in the position of the local maxima of the spectrum passing the sample in real time immediately and after 1 minute after filling with a mixture of a specific antigen and an assay solution containing the desired antibodies to this antigen. The shift in the position of the maxima linearly depends on the number of formed antigen-antibody complexes. To record the comparison spectra, radiation from a white light source is used, which is fed through a fiber-optic cable to a microlens mounted on a three-coordinate adjustment slide. With the help of a microlens, the radiation is focused and a small-diameter focal spot is created to enter the radiation strictly into the hollow core of the SMV PS sample.

The inventive method is carried out as follows.

SMV PS is placed in a special disposable cuvette, which is positioned with the help of a three-coordinate adjustment slide so that the end of the waveguide is exactly in the focus of the microlens. Thus, the radiation beam is injected into the hollow core of the waveguide. The radiation emerging from the SMV PS hollow core is collected by the second microlens and fed to the input of the fiber-optic cable of the spectrum analyzer, which is directly connected to the computer.

Then, specific binding substances are introduced into the structural shell of the SMV PS: an aqueous solution of antigen at a concentration of 5-15 μg / ml and a solution of the analyzed blood serum. For the quantitative determination of specific antibodies in standard immunological methods, a geometric series of analyte dilutions (blood sera) is used. The optimal dilution of serum, containing specific antibodies, is on average 1: 100-1: 800 [Immunological methods / ed. G. Frimel, trans. with him. A.P. Tarasova. 1987. M .: Medicine. 472 s.].

The optical spectrum probes the binding of analytes in the bulk of the structural shell of the SMV PS, which form a variety of analyte-sensitive devices. Thus, the area (volume) of measurement and the throughput of the immunosensor based on the SSM PS are increased. As the antigen-antibody complexes are formed, new positions of the local maxima of the transmission spectrum of the modified sample are measured and a linear dependence of the position of the local maximum on the number of formed antigen-antibody complexes is carried out. A typical transmittance spectrum of an MSWS is shown in FIG. 1. The transmission peaks of the obtained spectrum are transformed and shifted to long-wavelength or short-wavelength region, changing their intensity as the antigen-antibody complexes are formed (Fig. 2). In the structural envelope, the analyzed antibodies, if they are contained in the test samples, selectively bind to the analyte-specific antigenic molecules, as a result of which a shift or decrease in the wavelength occurs, which leads to a change in the position and shape of the local maxima of the band in the transmission spectrum of the CMV PS sample (Fig. . 3). In control samples that do not contain the analyzed antibodies, a specific reaction does not occur and changes in the shape of local maxima in the transmission spectrum of the sample CMB PS are not detected (Fig. 4). The position and intensity of the maxima are influenced by several characteristics of the substance placed in the structural shell, namely, the refractive index, absorption, scattering, and transmission of the waveguide itself. With the formation of antigen-antibody complexes, first of all, the scattering of radiation in the hollow core of the CMB begins to increase due to the fact that the complex molecule increases in size and, in accordance with this, the intensity begins to decrease. This is clearly illustrated in FIG. 3 and FIG. 4. In addition, the characteristic peaks of the empty waveguide are transformed due to the change in the refractive index of the core. So, in an empty waveguide, the channels are a priori filled with air with a refractive index of 1, when a liquid, such as water, enters, the refractive index of 1.33 of the number of peaks decreases and their half-width increases (blue, free-standing curve in Fig. 3 and Fig. 4).

Example 1.

To implement the method for detecting antibodies in the test samples, according to the claimed invention, an aqueous solution of antigen molecules is taken (an aqueous solution of F1 protein from the Yersinia pestis EV vaccine strain of the NIIEG line, at a concentration of 5 μg / ml), mixed with the test samples (in this example, these are experimental serum samples from people immunized with the plague live vaccine (HPL), as well as control serum samples from people in the control group who have not previously been vaccinated against plague and have not had contact with the causative agent or vector of this disease), diluted with phosphate buffered saline (PBS) in the ratio 1: 320. The analyzed antibodies, if they are present in the specified sample, interact with the specified antigen with the formation of complexes containing the antigenic molecule - the analyzed antibody. To detect the presence of these complexes, to obtain an indicator of the presence of the analyzed antibodies in the specified sample, it is introduced into the structural shell of the SMV PS. Detection is carried out in real time without the use of means for labeling the formed antigen-antibody complexes, using radiation (from a broadband source, LED, supercontinuum source) connected to the SMV PS. In the structural envelope of the SMV PS, the analyzed antibodies, if they are contained in the test samples, selectively bind to the analyte-specific antigenic molecules, as a result of which the position and shape of the local maxima in the transmission spectrum of the CMV PS sample change (Fig. 3). In control samples that do not contain the analyzed antibodies, a specific reaction does not occur and changes in the shape of local maxima in the transmission spectrum of the sample CMB PS are not detected (Fig. 4).

Example 2.

The specificity of the method for detecting antibodies in a test sample, according to the invention, was determined by the interaction of antigen molecules with diagnostic sera against antigens of plague, cholera and tularemia. For this, a mixture of antigen molecules (an aqueous solution of F1 protein from the Yersinia pestis EV strain at a concentration of 5 μg / ml) and a preparation of one of these sera, diluted with PBS to a diagnostic titer according to the instructions, was introduced into the structural shell of the CMB PS. The detection was carried out in real time, without the use of means for labeling the formed antigen-antibody complexes, using radiation (from a broadband source, LED, supercontinuum source) connected to the SMV PS. The specificity of the method was confirmed by the detection of antibodies only in a mixture of antigen molecules with a preparation of diagnostic serum against plague antigens. In the structural envelope of the SMV PS, the analyzed antibodies contained in the diagnostic serum against plague antigens selectively bind to the F1 protein molecules, as evidenced by the change in the position of the maxima, the change in the shape of the spectrum, and a drop in the transmission intensity of the CMB PS sample filled with the specified mixture (Fig. 5). In the samples of SMV PS containing a mixture of F1 protein molecules and a preparation of serum against cholera antigens, or a preparation against tularemia antigens that do not contain the analyzed antibodies, there was no specific reaction and no change in the position of the maxima, changes in the shape of the spectrum and a drop in the transmission intensity of the sample of SMV PS did not occur ( Fig. 6, 7).

The method according to the invention can be carried out partially manually or automatically. The detection of antibodies according to the invention can be partially automated using suitable sensor equipment and / or computerized processing and / or evaluation of the primary signals.

Comparative analysis and advantages of the proposed method with the most common modern immunological methods for detecting antibodies are shown in the table.

table

Claims (1)

A method for detecting antibodies in a biomaterial using glass microstructure waveguides is characterized by the fact that it provides for the production of an optical immunosensor by introducing a reaction mixture of an analyzed sample with an antigen into the hollow core of a glass microstructure waveguide, followed by determination of antibodies by the position of local maxima of the transmission spectrum of the sample, in real time, up to and after filling with a mixture of antigen and an assay solution containing the desired antibodies to this antigen.

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